Advances in genomics technology and genetic engineering enabled the development of a new field, synthetic biology, which seeks to create novel behaviors in cells by introducing new genetic elements and integrating such elements into circuits to produce more complex behaviors. Unfortunately, due to the added complexity of mammalian gene regulation, circuit construction, and circuit integration, much research in synthetic biology has been limited to simpler organisms like bacteria and yeast. However, due to the great potential of mammalian synthetic biology for therapeutics and diagnostics, it is imperative that more effort be focused towards this area. One basic need for synthetic biology is orthogonal transcription machinery which can be used to finely control gene expression. I propose using advanced genome engineering techniques, such as Trackable Multiplex Recombineering (TRMR) and Protein Sequence Activity Relationship (ProSAR), to engineer orthogonal RNA polymerase/promoter pairs. The bacteriophage T7 RNA polymerase/promoter system is an excellent starting point for this project research due to its rapid transcription spee, low termination rate and high specificity for its promoter sequence. I will rationally design oligo to search the protein space to determine 'hot spots' that effect promoter binding and then subject these areas to saturation mutagenesis. Combinatorial mutations will also be explored via multiplex automated genome engineering techniques. I will characterize the engineered RNA polymerase/promoter pairs to determine strength of promoter and orthogonality. Ultimately, these fully characterized RNA polymerase/promoter systems can then be used as parts for genetic circuit design and integration in mammalian cells.